Stefanie B. Flohé

Invited review: Deterioration of the immune system after trauma: signals and cellular mechanisms

Multiple trauma leads to a deterioration of the immune system. On the one hand, hyperinflammation mediates remote organ damage and may lead to multi-organ failure. On the other hand, immunosuppression develops and promotes an enhanced risk to acquire infectious complications after trauma. The mechanisms that underlie these opposing consequences of trauma are not yet completely understood. There is increasing evidence that endogenous danger signals that derive from destroyed tissues play a role in trauma-induced immune dysfunction. Here, we give an overview on the common animal models that are used to investigate trauma-induced pathology, potential signals and cellular mechanisms that support the imbalance between inflammation and counter-regulation after trauma.

A wheat-based, diabetes-promoting diet induces a Th1-type cytokine bias in the gut of NOD mice

Dietary antigens are candidate environmental factors in the pathogenesis of type 1 diabetes. In the non-obese diabetic (NOD) mouse, an animal model of type 1 diabetes, cereal-based diets promote disease development, whereas the diets based on hydrolysed proteins or non-diabetogenic proteins are protective. The hypothesis that diabetogenic diets modulate the cytokine balance in the gut was tested. NOD mice were fed with NTP-2000 (mainly a wheat-based milk-free diet) or Prosobee (a semi-purified hypoallergenic diet based on soy protein isolate) or Prosobee plus casein (milk protein fraction). The mRNA levels of IFN-gamma, IL-10, TNF-alpha, TGF-beta, and inducible NO synthase in the small intestine and the Peyers patches were determined by semi-quantitative RT-PCR. Mice fed on the cereal-based NTP-2000 diet expressed higher levels of the Th1-type and pro-inflammatory markers IFN-gamma, TNF-alpha, and inducible NO synthase mRNA compared to the Prosobee-fed animals. The expression of the counterregulatory cytokines IL-10 and TGF-beta was unaffected. This resulted in a significant bias of the intestinal cytokine balance towards T helper cell type 1 after feeding NTP-2000. The cytokine mRNA levels in the gut-associated Peyers patches were not affected. Thus, modulation of gut immunoreactivity by diet may contribute to disease development in NOD mice.

Modulation of Dendritic Cell Differentiation in the Bone Marrow Mediates Sustained Immunosuppression after Polymicrobial Sepsis

Murine polymicrobial sepsis is associated with a sustained reduction of dendritic cell (DC) numbers in lymphoid organs and with a dysfunction of DC that is considered to mediate the chronic susceptibility of post-septic mice to secondary infections. We investigated whether polymicrobial sepsis triggered an altered de novo formation and/or differentiation of DC in the bone marrow. BrdU labeling experiments indicated that polymicrobial sepsis did not affect the formation of splenic DC. DC that differentiated from bone marrow (bone marrow-derived DC [BMDC]) of post-septic mice released enhanced levels of IL-10 but did not show an altered phenotype in comparison with BMDC from sham mice. Adoptive transfer experiments of BMDC into naive mice revealed that BMDC from post-septic mice impaired Th1 priming but not Th cell expansion and suppressed the innate immune defense mechanisms against Pseudomonas bacteria in the lung. Accordingly, BMDC from post-septic mice inhibited the release of IFN-γ from NK cells that are critical for the protection against Pseudomonas. Additionally, sepsis was associated with a loss of resident DC in the bone marrow. Depletion of resident DC from bone marrow of sham mice led to the differentiation of BMDC that were impaired in Th1 priming similar to BMDC from post-septic mice. Thus, in response to polymicrobial sepsis, DC precursor cells in the bone marrow developed into regulatory DC that impaired Th1 priming and NK cell activity and mediated immunosuppression. The absence of resident DC in the bone marrow after sepsis might have contributed to the modulation of DC differentiation.

Origin of immunomodulation after soft tissue trauma: potential involvement of extracellular heat-shock proteins.

Severe injury may lead to immunosuppression, multiple organ failure, and death. The aim of the study was to investigate the direct impact of soft tissue destruction on the development of trauma-associated immunomodulation. Hip surgery was considered to represent an isolated soft tissue trauma that allowed for the examination of changes taking place locally at the site of trauma or systemically with regard to monocyte function and leukocyte redistribution. Peripheral blood and wound fluid collected from the drains of 21 patients after hip surgery were analyzed to determine the cellular composition and/or the responsiveness of mononuclear cells (MNCs) to lipopolysaccharide (LPS). Different factors present in the wound fluids were tested for their capacity to modulate the MNC of healthy individuals with regard to cytokine and chemokine secretion. We found that various factors, including heat-shock protein (HSP) 60 and HSP70, were locally released at the site of soft tissue trauma and could be detected in wound fluids. The wound fluid-derived MNC (but not the peripheral blood-derived MNC) showed an impaired capacity to release TNF-&agr; after LPS stimulation. Cell-free wound fluid suppressed in healthy individuals the LPS-induced TNF-&agr; secretion by MNC. After surgery, granulocytosis was found in peripheral blood and in wound fluids, but monocytopenia was restricted to wound fluids. In parallel, wound fluids induced in healthy individuals the release by MNC of distinct chemokines specific for granulocytes and monocytes. These wound fluid-mediated effects of TNF-&agr; suppression and chemokine induction could be mimicked by recombinant human HSP70 and, in part, by HSP60. Thus, tissue-derived factors, such as HSP70 released after injury, suppress monocyte function and, therefore, might favor the development of immunosuppression after severe injury.ABBREVIATIONS-IL-interleukin; LPS-lipopolysaccharide; TNF-tumor-necrosis factor; MCP-1-monocytes-chemoattractant protein; RANTES-regulated on activation normal T expressed and secreted; HSP-heat-shock protein; MNC-mononuclear cells; PB-MNC-peripheral blood-derived MNC; WF-MNC-wound fluid-derived MNC

Mesenchymal stem cells augment the anti-bacterial activity of neutrophil granulocytes.

Background Mesenchymal stem cells (MSCs) participate in the regulation of inflammation and innate immunity, for example by responding to pathogen-derived signals and by regulating the function of innate immune cells. MSCs from the bone-marrow and peripheral tissues share common basic cell-biological functions. However, it is unknown whether these MSCs exhibit different responses to microbial challenge and whether this response subsequently modulates the regulation of inflammatory cells by MSCs. Methodology/Principal Findings We isolated MSCs from human bone-marrow (bmMSCs) and human salivary gland (pgMSCs). Expression levels of TLR4 and LPS-responsive molecules were determined by flow cytometry and quantitative PCR. Cytokine release was determined by ELISA. The effect of supernatants from unstimulated and LPS-stimulated MSCs on recruitment, cytokine secretion, bacterial clearance and oxidative burst of polymorphonuclear neutrophil granulocytes (PMN) was tested in vitro. Despite minor quantitative differences, bmMSCs and pgMSCs showed a similar cell biological response to bacterial endotoxin. Both types of MSCs augmented anti-microbial functions of PMNs LPS stimulation, particularly of bmMSCs, further augmented MSC-mediated activation of PMN. Conclusions/Significance This study suggests that MSCs may contribute to the resolution of infection and inflammation by promoting the anti-microbial activity of PMNs. This property is exerted by MSCs derived from both the bone-marrow and peripheral glandular tissue.

Interaction with Mesenchymal Stem Cells Provokes Natural Killer Cells for Enhanced IL-12/IL-18-Induced Interferon-Gamma Secretion

Tissue injury induces an inflammatory response accompanied by the recruitment of immune cells and of mesenchymal stem cells (MSC) that contribute to tissue regeneration. After stimulation with interleukin- (IL-) 12 and IL-18 natural killer (NK) cells secrete the proinflammatory cytokine interferon- (IFN-) γ. IFN-γ plays a crucial role in the defense against infections and modulates tissue regeneration. In consideration of close proximity of NK cells and MSC at the site of injury we investigated if MSC could influence the ability of NK-cells to produce IFN-γ. Coculture experiments were performed with bone marrow-derived human MSC and human NK cells. MSC enhanced the ability of IL-12/IL-18-stimulated NK cells to secrete IFN-γ in a dose-dependent manner. This activation of NK cells was dependent on cell-cell contact as well as on soluble factors. The increased IFN-γ secretion from NK cells after contact with MSC correlated with an increased level of intracellular IFN-γ. Alterations in the IL-12 signaling pathway including an increased expression of the IL-12β1 receptor subunit and an increased phosphorylation of signal transducer and activator of transcription 4 (STAT4) could be observed. In conclusion, MSC enhance the IFN-γ release from NK cells which might improve the defense against infections at the site of injury but additionally might affect tissue regeneration.

Human mesenchymal stromal/stem cells acquire immunostimulatory capacity upon cross-talk with natural killer cells and might improve the NK cell function of immunocompromised patients

BackgroundThe suppressive effect of mesenchymal stromal/stem cells (MSCs) on diverse immune cells is well known, but it is unclear whether MSCs additionally possess immunostimulatory properties. We investigated the impact of human MSCs on the responsiveness of primary natural killer (NK) cells in terms of cytokine secretion.MethodsHuman MSCs were generated from bone marrow and nasal mucosa. NK cells were isolated from peripheral blood of healthy volunteers or of immunocompromised patients after severe injury. NK cells were cultured with MSCs or with MSC-derived conditioned media in the absence or presence of IL-12 and IL-18. C-C chemokine receptor (CCR) 2, C-C chemokine ligand (CCL) 2, and the interferon (IFN)-γ receptor was blocked by specific inhibitors or antibodies. The synthesis of IFN-γ and CCL2 was determined.ResultsIn the absence of exogenous cytokines, trace amounts of NK cell-derived IFN-γ licensed MSCs for enhanced synthesis of CCL2. In turn, MSCs primed NK cells for increased release of IFN-γ in response to IL-12 and IL-18. Priming of NK cells by MSCs occurred in a cell–cell contact-independent manner and was impaired by inhibition of the CCR2, the receptor of CCL2, on NK cells. CD56bright NK cells expressed higher levels of CCR2 and were more sensitive to CCL2-mediated priming by MSCs and by recombinant CCR2 ligands than cytotoxic CD56dim NK cells. NK cells from severely injured patients were impaired in cytokine-induced IFN-γ synthesis. Co-culture with MSCs or with conditioned media from MSCs and MSC/NK cell co-cultures from healthy donors improved the IFN-γ production of the patients’ NK cells in a CCR2-dependent manner.ConclusionsA positive feedback loop driven by NK cell-derived IFN-γ and MSC-derived CCL2 increases the inflammatory response of cytokine-stimulated NK cells not only from healthy donors but also from immunocompromised patients. Therapeutic application of MSCs or their soluble factors might thus improve the NK function after severe injury.

Lipid-rich enteral nutrition improves the defense against an opportunistic infection during polymicrobial sepsis

ABSTRACT The development of an immunosuppressive state during the protracted course of sepsis is associated with opportunistic infections and is considered to correlate with the extent of the proinflammatory response during early sepsis. Short-term intervention with enteral lipid-rich nutrition was shown to attenuate the acute inflammatory response. This study investigates the effects of lipid-rich nutrition on the immunosuppression induced by polymicrobial sepsis. Female BALB/c mice were either fasted or fed liquid lipid-rich nutrition or isocaloric control nutrition before and shortly after induction of polymicrobial sepsis through cecal ligation and puncture (CLP) or sham operation. After 4 days, mice were intranasally infected with Pseudomonas aeruginosa. Twenty-four hours after P. aeruginosa infection, fasted and control nutrition-fed CLP mice displayed a significantly higher bacterial load in the lungs than did corresponding sham-operated mice (P < 0.001 and P < 0.05, respectively). Fasted CLP mice expressed reduced pulmonary levels of proinflammatory cytokines interleukin 12 (IL-12) and interferon &ggr; (IFN-&ggr;) in comparison to sham mice (both P < 0.05). Lipid-rich nutrition prevented the increase in bacteria, promoted the IL-12 and IFN-&ggr; production (IL-12 and IFN-&ggr; [P < 0.05] vs. fasted and IFN-&ggr; [P < 0.05] vs. control nutrition), and prevented the expression of the immunosuppressive cytokine IL-10 (P < 0.05 vs. control nutrition) in lungs of CLP mice. The preserved immune defense during late sepsis in lipid-rich fed mice was preceded by attenuation of the early inflammatory response (IL-6 [P = 0.05] and IL-10 [P < 0.01] vs. fasted CLP mice) at 6 h after CLP. In conclusion, short-term treatment with lipid-rich enteral nutrition improves the pulmonary antimicrobial defense during polymicrobial sepsis.

ACTIVATION OF HYPOXIA-INDUCIBLE FACTOR 1 IN SKELETAL MUSCLE CELLS AFTER EXPOSURE TO DAMAGED MUSCLE CELL DEBRIS

Skeletal muscle damage provokes complex repair mechanisms including recruitment of leukocytes as well as activation of myogenic precursor cells such as satellite cells. To study muscle cell repair mechanisms after muscle fiber damage, we used an in vitro model of scrape-injured myotubes. Exposing vital C2C12 myoblasts and myotubes to cell debris of damaged myotubes revealed mRNA upregulation of adrenomedullin (ADM), insulin-like growth factors 1 and 2, metallopeptidase 9, and monocyte chemoattractant protein11. When cell debris was treated with ultrasound, frozen in liquid nitrogen, or heat inactivated before addition to C2C12 cells, gene expression was drastically reduced or completely absent. Moreover, incubations of myoblasts with debris separated by transwell inserts indicated that direct cell contact is required for gene induction. Incubation with albumin and PolyIC ruled out that ADM induction by cell debris simply results from increased protein or nucleic acid concentrations in the supernatant. Because the genes, which were upregulated by cell debris, are potential target genes of hypoxia-inducible factor (HIF), cells were analyzed for HIF-1&agr; expression. Western blot analysis showed accumulation of the &agr;-subunit upon contact to cell debris. Knockdown of HIF-1&agr; in C2C12 cells proved that activation of HIF-1 in response to cell debris was responsible for upregulating ADM and monocyte chemoattractant protein 1. Furthermore, by incubating cells on gas-permeable culture dishes, we excluded a reduced pericellular pO2 induced by cell debris as the cause for ADM upregulation. Our data suggest that damaged myofibers activate HIF-1 in neighboring myotubes and precursor myoblasts by direct contact, concomitantly upregulating factors necessary for angiogenesis, tissue regeneration, and phagocyte recruitment.ABBREVIATIONS-ADM-adrenomedullin; HIF-hypoxia-inducible factor; IGF-insulin-like growth factor; IFN-interferon; IRAK-IL-1 receptor-associated kinase; LPS-lipopolysaccharide; MMP-metallopeptidase; MCP-monocyte chemoattractant protein; SOD-superoxide dismutase; TLR-toll-like receptor

Invasive Surgery Impairs the Regulatory Function of Human CD56bright Natural Killer Cells in Response to Staphylococcus aureus. Suppression of Interferon-γ Synthesis

Major surgery increases the risk for infectious complications due to the development of immunosuppression. CD56bright NK cells play a key role in the defense against bacterial infections through the release of Interferon (IFN) γ upon stimulation with monocyte-derived Interleukin (IL) 12. We investigated whether invasive visceral surgery interferes with the IFN-γ synthesis of human NK cells in response to Staphylococcus aureus. In a prospective pilot study, peripheral blood mononuclear cells (PBMC) were isolated from 53 patients before and 1 to 7 d after elective visceral surgery. The release of IL-12 and IFN-γ from PBMC upon exposure to S. aureus in vitro was quantified. The expression of the IL-12 receptor β1 chain on the surface, the phosphorylation of signal transducer and activator of transcription (STAT) 4, and the synthesis of IFN-γ on/in individual CD56bright NK cells were investigated using flow cytometry. The modulatory effect of IL-12 on the S. aureus-induced IFN-γ production in CD56bright NK cells was analyzed. The IFN-γ secretion from purified CD56bright NK cells was quantified after stimulation with IL-12 and IL-18. After surgery, CD56bright NK cells among total PBMC were impaired in the release of IFN-γ for at least 5 d. Likewise, the IL-12-induced release of IFN-γ from purified CD56bright NK cells was abolished. Upon stimulation with S. aureus, PBMC secreted less IL-12 but supplementation with recombinant IL-12 did not restore the capacity of CD56bright NK cells to produce IFN-γ. CD56bright NK cells displayed reduced levels of the IL-12Rβ1 chain whereas the phosphorylation of STAT4, the key transcription factor for the Ifng gene was not diminished. In summary, after invasive visceral surgery, CD56bright NK cells are impaired in S. aureus-induced IFN-γ production and might contribute to the enhanced susceptibility to opportunistic infections.